A Parallel G Quadruplex-Binding Protein Regulates the Boundaries of DNA Elimination Events of Tetrahymena thermophila

Abstract

Guanine (G)-rich DNA readily forms four-stranded quadruplexes in vitro, but evidence for their participation in genome regulation is limited. We have identified a quadruplex-binding protein, Lia3, that controls the boundaries of germline-limited, internal eliminated sequences (IESs) of Tetrahymena thermophila. Differentiation of this ciliate's somatic genome requires excision of thousands of IESs, targeted for removal by small-RNA-directed heterochromatin formation. In cells lacking LIA3 (ΔLIA3), the excision of IESs bounded by specific G-rich polypurine tracts was impaired and imprecise, whereas the removal of IESs without such controlling sequences was unaffected. We found that oligonucleotides containing these polypurine tracts formed parallel G-quadruplex structures that are specifically bound by Lia3. The discovery that Lia3 binds G-quadruplex DNA and controls the accuracy of DNA elimination at loci with specific G-tracts uncovers an unrecognized potential of quadruplex structures to regulate chromosome organization.

Fig 1. ΔLIA3 matings have reduced progeny production.

(A) rt-pcr of vegetatively growing (G), starved (S), and conjugating cells (3, 6, 9, and 12 hrs postmixing). HhpI is used as a loading control. Black arrows point to cDNA band; grey arrows point to DNA band. Reverse transcriptase was omitted when synthesizing the cDNA for the bottom gels to confirm lack of gDNA contamination in RNA samples. (B) Percent of cells that either complete conjugation by resorbing one of their micronuclei or do not complete conjugation and maintain two micronuclei. (C) Percent of mated pairs that produce viable progeny for each mating. N > 200 for WT x WT and ΔLIA3 x ΔLIA3. N > 45 for WT x ΔLIA3 matings. For b and c, ΔLIA1 mating data from [58].

Fig 2. Excision of the M IES is aberrant and delayed in ΔLIA3 matings.

(a and d) Diagram illustrating the alternative rearranged forms of the M IES (a) or R IES d). Grey boxes represent the macronucleus retained flanking regions; white boxes, the 0.6 kb and 0.3 kb eliminated sequences; asterisks, the A5G5 tracts; arrows, the location of the pcr primers used in b and c to amplify across the IES. (b and e) PCR of genomic DNA isolated from different timepoints throughout mating demonstrating that excision of the M IES (b) is both delayed and aberrant in ΔLIA3 matings while excision of the R IES is normal (e). (c and f) PCR of individual progeny confirming that excision of the M IES (c) is aberrant in ΔLIA3 matings whereas excision of the R IES (f) is comparable to WT matings. Black arrows point to the unrearranged form and single and double grey arrows point to the expected rearranged forms for b, c, e, and f. In b and c, the doublet observed for the 0.6 kbp M IES deletion reveals that some fraction of wild-type excision events are directed by a cryptic A5G5 tract (AAAGGAGG) rather than the major A5G5 boundary determinant. (g) Diagram displaying the sequenced junctions from individual ΔLIA3 progeny. A diagram of the M IES denotes the two alterative left boundaries, M1 and M2, and the right boundary M3. Within the IES diagram grey boxes represent the flanking regions; white boxes, the 0.6 kb and 0.3 kb eliminated region of the M IES; asterisks, A₅G₅ tracts; arrows, pcr primers. Each thin, white and black boxes beneath the IES diagram represents the region excised from the progeny of either wt or ΔLIA3 matings, respectively. (h) The strategy to detect excised IES circles: PCR primers located within each IES point outward toward the boundaries and will only amplify a product if the excised region forms a circular intermediate. Grey boxes represent the flanking region; white boxes, the excised region; arrows, pcr primers. (i) PCR results for the excised M IES throughout mating. Primers will not amplify circular products utilizing the interior, right deletion boundary. Note: the lower band in the 14 hr WT mating sample corresponds to an alternative form seen multiple times in WT matings. (j) PCR results for the excised R IES throughout mating. Arrows in i and j point to the expected size of the circular product. (k) Diagram of the excised regions based on sequencing the circular intermediates from Fig 2j.

Fig 3. Lia3 acts on the flanking region of the M IES.

(a and e) Schematic of the expected rearrangement in wt cells of chimeric IESs used in the Southern blots shown in b, c, e, and f. Boxes labeled M or R represent the flanking DNA of the M IES or R IES; light grey boxes, the 0.3kb internal region of the M IES; box labeleled M IES or R IES or TLR IES, the indicated germline-limited sequence; black bar, Southern probe (b-d, f-h). Each lane of the Southern blot contains genomic DNA isolated from three co-cultured transformants. The diagram above each Southern blot denotes which chimeric IES-rDNA vector was introduced into mating cells. Black arrows indicate the position of the unrearranged DNA, while grey arrows point to fragments of the size expected after accurate IES excision.

Fig 4. ΔLIA3 lines aberrantly rearrange IES flanked by A₅G₅.

Lanes 1–4 are from the wt and ΔLIA3 parent lines used for the matings in lanes 5–13. (a-c) PCR across rearrangement junction of IES flanked by A₅G₅ that display aberrant rearrangement in ΔLIA3 matings. (d-f) PCR across rearrangement junction of IES not flanked by A₅G₅ that rearrange normally in ΔLIA3 matings. Black arrows, pcr primers; asterisks, polypurine tracts; grey boxes, flanking sequences; white boxes, IES. IES 55 has a sequence resembling a polypurine tract only on its right flank.

Fig 5. Lia3 binds M IES flanking DNA, which adopts a quadruplex structure.

(a) Competition gel shift in which 300 nM His-Lia3 was incubated with 5 times as much unlabeled competitor oligo (listed on the left) followed by incubation with ³²P-labeled oligo (listed above) before loading on a 4% native gel. (b) Schematic representation of a G4 DNA structure. (c) CD spectrum of the M1 oligo in 100 mM KCl buffer.

Fig 6. Lia3 binds a parallel G-quadruplex forming oligonucleotide in vitro.

(a) 50–400 nM MBP-Lia3 was incubated with ssM1 (LiCl) or G4 M1 (KCl) probes and binding was analyzed on 5% polyacrylamide gels. Increasing amounts of either wt M1 or C3G2 mutant M1 oligonucleotide was added to assess competition in the presence of 400 nM MBP-Lia3. (b) Binding curves as determined by EMSA for His-Lia3 binding to ssM1, dsM1, G4-M1, and G4-Tetrahymena telomere.

Fig 7. Possible model for how Lia3 determines IES boundaries.

(A) RT-PCR across the A₅G₅ located at the left boundary of the M IES at 3, 6, 9, or 12 hours post-mixing. (B) Model: Shortly after the new macronuclei form, Twi1 bound to scnRNAs interacts with the nascent transcripts being produced across IES. This interaction recruits the histone methyltransferase Ezl1 to induce H3K9/27me at the IES. After H3K9me3 and H3K27me3 marks have been placed, Pdd1, Pdd3 and other chromodomain containing proteins recognize these marks and induce bending in the DNA causing the two ends of the IES to move towards each other. Around this time, transcription is occurring throughout the developing nuclei including across the polypurine tracts located near the boundaries. This opens up the double helix and allows the single stranded DNA to bind to the newly synthesized messenger RNA. Since the two ends are now in proximity to each other, the RNA/DNA hybrids on each side can bind to each other and form a quadruplex. Lia3 binding to the quadruplex either stabilizes the quadruplex interaction thereby keeping the two ends locked together and facilitating Tpb2 in cutting at the correct location or Lia3 recruits Tpb2 to the correct location through a direct interaction. Dotted lines indicate newly synthesized RNA; solid lines, DNA; hexagons, methylation marks; cylinders, nucleosomes.